Substrate Recognition Governs Reverse Transcriptase Resistance to Diagnostic Inhibitors in RT-qPCR

Background: Reverse transcription is a key step in emerging RNA diagnostics, but reverse transcriptase (RT) enzymes often fail in the presence of inhibitors carried over from clinical samples or introduced during RNA extraction. Here, we dissect the molecular basis of inhibitor resistance in five engineered variants (V1 to V5) of Moloney Murine Leukemia Virus RT, originally optimized for thermostability and catalytic activity. Methods: Using a systematic framework that integrates structural analysis, thermal profiling, and diagnostic benchmarking, we evaluated cDNA synthesis from 40 to 70 °C under a panel of 11 clinically relevant inhibitors. Results: Across 30 mutations assessed, a recurrent set of substitutions (E69K, E302K/R, W313F, and N454K), present in RT V1 and V4, was associated with enhanced robustness, consistent with strengthened enzyme–nucleic acid engagement, while L435G likely contributes by modulating conformational flexibility. Notably, inhibitor tolerance was maximal at moderate reaction temperatures (≈40 °C), where productive enzyme–substrate interactions best offset inhibitory stress, while the wild-type enzyme was effectively inactivated by several inhibitors under the conditions tested. Although the engineered RTs remained catalytically competent at higher temperatures, increased thermal stress may destabilize productive enzyme–nucleic acid complexes, reducing resistance under inhibitory conditions. Conclusions: Together, these findings support substrate engagement as an important determinant of RT robustness and provide practical guidance for engineering inhibitor-resistant RTs for high-sensitivity RT-qPCR.